Panax notoginseng: panoramagram of phytochemical and pharmacological properties, biosynthesis, and regulation and production of ginsenosides

Abstract Panax notoginseng is a famous perennial herb widely used as material for medicine and health-care food. Due to its various therapeutic effects, research work on P. notoginseng has rapidly increased in recent years, urging a comprehensive review of research progress on this important medicinal plant. Here, we summarize the latest studies on the representative bioactive constituents of P. notoginseng and their multiple pharmacological effects, like cardiovascular protection, anti-tumor, and immunomodulatory activities. More importantly, we emphasize the biosynthesis and regulation of ginsenosides, which are the main bioactive ingredients of P. notoginseng. Key enzymes and transcription factors (TFs) involved in the biosynthesis of ginsenosides are reviewed, including diverse CYP450s, UGTs, bHLH, and ERF TFs. We also construct a transcriptional regulatory network based on multi-omics data and predicted candidate TFs mediating the biosynthesis of ginsenosides. Finally, the current three major biotechnological approaches for ginsenoside production are highlighted. This review covers advances in the past decades, providing insights into quality evaluation and perspectives for the rational utilization and development of P. notoginseng resources. Modern omics technologies facilitate the exploration of the molecular mechanisms of ginsenoside biosynthesis, which is crucial to the breeding of novel P. notoginseng varieties. The identification of functional enzymes for biosynthesizing ginsenosides will lead to the formulation of potential strategies for the efficient and large-scale production of specific ginsenosides.


Introduction
Panax notoginseng (Burk.)F.H. Chen, also known as Sanchi, is a perennial herb belonging to the Araliaceae family, extensively detailed in the classical Chinese pharmacopeia, Ben-Cao-Gang-Mu [1].Originating from East Asia and North America around 25 million years ago, it predominantly thrives in China's southwestern Yunnan and Guangxi regions, with the Yunnan variant highly regarded for its medicinal properties [2].As both a medicinal and edible plant, P. notoginseng has garnered considerable interest for its applications in pharmaceuticals, functional foods, and health-care products [3].Its notable effectiveness in enhancing blood circulation and alleviating pain renders it crucial in cardiovascular disease treatments, featuring prominently in traditional Chinese medicine prescriptions such as Yunnan Baiyao, Pientzehuang Compound, Danshen Dripping Pills, and Xuesaitong Injection [4].Beyond its medical uses, P. notoginseng is prized for its health benefits and culinary applications.Its f lowers are brewed into herbal teas that help cool the body and lower blood pressure, while its stems and leaves are utilized in coughrelieving teas.The roots, known for their anti-inf lammatory and cardioprotective attributes, are often added to soups [5].With its widespread popularity in various commercial sectors, including health and beauty, and dietary supplements, P. notoginseng enjoys broad recognition across Asia and in the West [6].Responding to increasing demand, the industry has seen significant growth, with production reaching 28 000 tons and generating annual revenues of 16.2 billion Chinese yuan in 2017 [7].
The metabolites of P. notoginseng play a crucial role in its pharmacological efficacy.This plant harbors a diverse array of secondary metabolites, including ginsenosides, organic acids, esters, polysaccharides, amino acids, sterols, and f lavonoids.These ingredients have significant therapeutic effects on the nervous system and immune response [8].Ginsenosides stand out as the foremost bioactive elements, driving the medicinal effectiveness of this herb [9].As the demand for P. notoginseng escalates, so does the interest in its agricultural production and the enhancement of biosynthetic methods for these active compounds.Such advancements are vital for the progression of herbal medicine and resource conservation.Plant metabolites are synthesized, transported, and metabolized through complex metabolic networks.Despite this, there remains a notable gap in the comprehensive analysis of the biosynthesis of these crucial active ingredients and their transcriptional regulation networks.
This review focuses on four principal topics: (i) bioactive constituents of P. notoginseng, (ii) its pharmacological benefits, (iii) the biosynthesis and regulation of ginsenosides, and (iv) biotechnological approaches to ginsenoside production.An in-depth exploration of the structures, distributions, biosynthetic routes, transcriptional regulatory frameworks, and biotechnological production techniques of ginsenosides is indispensable for the qualitative assessment and optimal exploitation of P. notoginseng.

Bioactive ingredients
The therapeutic effects of P. notoginseng are mainly attributed to their bioactive components, such as saponins, amino acids, polysaccharides, and f lavonoids [10].Saponins are mainly divided into three dammarane-type tetracyclic triterpenoids, oleananetype pentacyclic triterpenoids, and ocotilloltype pentacyclic triterpenoids, including ginsenosides Rb1, Rc, Rb2, Rg1, Re, Ro, and Rd, notoginsenoside R1, and majonoside R2.Amino acids include a non-protein amino acid (dencichine) and protein amino acids (such as arginine, aspartic acid, glutamic acid, and leucine).Polysaccharides include polysaccharide I, II (IIa, IIb), and III categories, and are mainly made up of glucose, galactose, and arabinose.The basic parent nucleus of f lavonoids is 2phenylketonuria and the basic skeleton of f lavonoids is C6-C3-C6, and they mainly consist of quercetin, quercetin-7-glucoside, f lavonoside quercetin-3-O sophoroside, and kaempferic acid.The types and contents of these bioactive components can be inf luenced by region, tissue, and development age, as discussed below.
The distribution and concentration of individual ginsenosides vary across different plant parts and developmental stages, as shown in Fig. 1B [20].The total ginsenoside content in 3-yearold plants is 1.4 times higher than that in 2-year-old plants, with PPD-type ginsenosides predominantly distributed in aboveground parts and PPT-type ginsenosides mainly distributed in underground parts [21].Leaves are particularly rich in PPD-type ginsenosides, including Rb3, Rc, and Rb2, while the main roots harbor significant levels of PPT-type ginsenosides such as Rg1, Re, and N-R1 [22,23].

Amino acids
Dencichine (β-N-oxalo-L-α-β-diaminopropionic acid, β-ODAP, C5H8N2O5), a non-protein amino acid with strong hemostatic activity and strong neuroexcitatory toxicity, is a characteristic component of P. notoginseng [24] (Supplementary Data Fig.S1 and Supplementary Data Table S1).The concentration of dencichine varies depending on the age and part of the plant, with the highest levels found in the f lowers and leaves [24].Specifically, dencichine content in 3-year-old f lowers is 2.98%, which is 1.48 times greater than in 2-year-old f lowers at 2.01%.Additionally, the method of drying significantly inf luences dencichine levels, the highest yield being observed with a drying treatment at 50 • C and the lowest with freeze-drying at 40 • C [25].
Furthermore, P. notoginseng is rich in protein amino acids, including arginine, aspartic acid, glutamic acid, and leucine (Supplementary Data Table S1).The concentration and composition of these amino acids vary across different plant parts and environments [26,27].Notably, the f lowers possess the highest total amino acid content but lack arginine, whereas the roots are devoid of both arginine and methionine.To date, researchers have identified 19 amino acids in the roots of P. notoginseng [26].
The polysaccharide levels in P. notoginseng are significantly affected by factors such as geographical location, time of harvest, and specific plant parts.The highest levels of polysaccharides are typically found in April and the lowest in July.Geographically, Yanshan shows the highest levels of polysaccharides, in contrast to the lower levels found in Guangxi [33].Within the plant, the polysaccharide content varies, with ribs exhibiting the highest levels and stems and leaves the lowest [34].

Pharmacological activities
An increasing number of studies demonstrate that P. notoginseng has been widely used in the treatment of some chronic diseases, such as atherosclerosis, diabetes, cancer, and cardiovascular and cerebrovascular diseases [38].Panax notoginseng saponins (PNS) are the major active ingredient of P. notoginseng and plays an important role in the medical and health fields.All kinds of PNS medicinal preparations, such as Sanqi-Tongshu capsules, Xuesaitong injection and Xuesaitong capsules, have been extensively used clinically.Modern research reveals that PNS possess a range of biological properties, including cardioprotective, cerebrovascular protective, neuroprotective, anti-tumor, anti-inf lammatory, hemostatic, and anticoagulant effects.The following is an overview of these therapeutic effects and their relevant mechanisms (Table 1 and Fig. 2).

Cardioprotective effect
Cardiovascular diseases are the primary causes of mortality in developed nations, accounting for half of all deaths [62].Research has demonstrated that P. notoginseng has potent protective effects on myocardial cells and combats cardiovascular ailments through a variety of complex signaling pathways.For instance, notoginsenoside R1 is acknowledged as a promising therapeutic to inhibit restenosis following percutaneous transluminal angioplasty, by hindering the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway [39].Moreover, notoginsenoside Ft1 boosts the expression of vascular endothelial growth factor (VEGF), stimulated by HIF-1α, linked to the PI3K/Akt and Raf/MEK/ERK signaling pathways [40].Additionally, PNS and its main constituents, ginsenosides Rg1 and R1, are known to significantly enhance endothelial cell migration and angiogenesis following myocardial infarction through the phosphorylation of AMPK Thr 172 and CaMKII Thr 287 [41].

Cerebrovascular protective effect
Cerebrovascular diseases rank as significant causes of disability and the third leading cause of mortality in developed nations [63].
PNS have shown efficacy in mitigating neurological deficits, cerebral infarctions, edema, and neuronal death [42,43].For instance, ginsenoside Rd is noted for potentially reducing tau phosphorylation induced by cerebral ischemia through the PI3K/Akt/GSK-3β pathway, and for lessening damage to the blood-brain barrier via the NF-κB/MMP-9 pathway [44,45].Additionally, ginsenoside Rb1 inf luences the Akt/mTOR/PTEN signaling pathway, offering therapeutic benefits for various neurological conditions [46].Ginsenoside Rg1 also plays a role in preventing IR-induced neurological injuries by blocking NF-κB nuclear translocation and the phosphorylation of IκBα, and by lowering glutamate and aspartate levels [47].

Neuroprotective effect
The incidence of neurodegenerative disorders such as Alzheimer's and Parkinson's is on the rise in developed nations, a trend attributed to longer life expectancies [64,65].Research into the therapeutic benefits of ginsenosides for these conditions has identified potential mechanisms involving the SKN-1 signaling pathway and the expression of Alzheimer's disease-related cir-cRNAs [48].Ginsenosides Rg1 and Rg2 are noted for enhancing cognitive function and reducing hippocampal amyloid-β (Aβ) deposition through modulation of associated metabolic pathways [49].Additionally, ginsenoside Rg1 combats cognitive decline through its antioxidant and anti-inf lammatory actions mediated by the PI3K/Akt/GSK-3β pathway [50].Ginsenoside Rb1 is also  used in managing Huntington's disease, traumatic brain injuries, and ischemia [66], while ginsenoside Rd is known to mitigate excitatory toxicity, modulate nerve growth factors, and promote nerve regeneration [67].

Anti-tumor effect
Research has demonstrated that P. notoginseng has significant anti-tumor effects against several types of cancer, including liver cancer [8].PNS inf luence immune responses and curb tumor proliferation through various mechanisms.For example, they reduce the immunosuppression of Treg cells within the colorectal cancer environment by inhibiting the expression of IDO1, which is controlled by signal transducer and activator of transcription 1 (STAT1) [51].Rb1 curtails the production of tumor necrosis factorα (TNF-α)-induced MMP-9 by modulating the RNA-dependent protein kinase and the NF-κB signaling pathways [52].R1 boosts antihepatoma efficacy by activating the PI3K/Akt pathway and suppressing miR-21, clarifying its role in combating hepatocellular carcinoma [53].Rg3 decreases NHE1 expression by blocking the epidermal growth factor receptor pathway, including phosphorylated extracellular signal-regulated kinase and hypoxiainducible factor 1α in hepatocellular carcinoma [54].Additionally, ginsenoside Rg5 promotes apoptosis and autophagy by disrupting the PI3K/Akt signaling pathway [55].

Biosynthesis and regulation of saponins
The most important active ingredients of P. notoginseng that exert pharmacological effects are ginsenosides, a series of triterpenoid saponins.However, the resources of P. notoginseng are currently limited.In order to better solve the source problem of ginsenosides, we need to comprehend the biosynthesis and regulation of ginsenosides in P. notoginseng, providing more clues for the breeding of novel P. notoginseng varieties with high ginsenoside contents and potential biotechnological methods for efficient and largescale production of ginsenosides.The genes and transcription factors (TFs) involved in the ginsenoside biosynthesis pathways of P. notoginseng primarily govern the enzymes that facilitate the structurally diverse biosynthesis of ginsenosides.Additionally, environmental factors significantly inf luence ginsenoside production through interactions within hormonal signal-transcription regulatory networks.The biosynthesis and regulation of ginsenosides of P. notoginseng are reviewed below.

Biosynthesis of ginsenosides
Ginsenosides in the Panax genus and their biosynthesis have been reported previously [76].The biosynthesis of ginsenosides primarily encompasses three stages: formation of the ginsenoside skeleton, glycosome synthesis, and modification of the skeleton [76].The synthesis of ginsenosides predominantly occurs through the mevalonic acid (MVA) pathway in the cytoplasm and the methylerythritol phosphate (MEP) pathway in plastids, as illustrated in Fig. 3.
3-Hydroxy-3-methylglutaryl CoA reductase (HMGR) is a crucial rate-limiting enzyme in ginsenoside biosynthesis [80].The enhancement of PnHMGR expression leads to an increase in ginsenoside production in P. notoginseng [81].Key enzymes such as PnFPS, PnSS, PnSE1, PnSE2, and PnDS are involved in this process and exhibit organ-specific expression patterns, showing particularly high levels in the f lowers of 4-year-old P. notoginseng [82].PnSE1 is present in all organs but predominantly in f lowers, while PnSE2 shows relatively low expression [83].The overall ginsenoside content is largely dependent on the activity of farnesyl diphosphate synthase (FPS), followed by SS and DS, with the concentration of Rb1 specifically linked to the activities of HMGR and FPS [84].Research has demonstrated that the genes PnFPS, PnSS, PnSE1, PnSE2, and PnDS have tissue-specific expression patterns and show significantly higher levels in the f lowers and leaves compared with the roots and fibrils, being 5.2 times greater [21,85].

Cytochrome P450
After the initial formation of the basic ginsenoside skeleton, it is further transformed into ginsenosides with great diversity in structure and function in the Panax genus via hydroxylation by cytochrome P450 enzymes (CYPs) and glycosylation by uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs).CYP enzymes are part of large and functionally diverse gene families [79].The subfamily CYP716, belonging to the CYP85 clade, is a major contributor to the diversification of triterpenoid biosynthesis [86].Specifically, three CYP716s -CYP716A47 (protopanaxadiol synthase, PPDS), CYP716A53v2 (protopanaxatriol synthase, PPTS), and CYP716A52v2 (oleanolic acid synthase, OAS) -have been identified [87][88][89].Dammarenediol-II undergoes hydroxylation by CYP716A47 and CYP716A53v2 to form protopanaxadiol and protopanaxatriol in four common Panax species (P.notoginseng, P. ginseng, P. quinquefolius, and P. japonicus) [90].Distinct from CYP716A47 and CYP716A53v2, CYP716A52v2 serves as a multifunctional oxygenase involved in the biosynthesis of oleanolic acid in P. ginseng, P. quinquefolius, and P. japonicus, but is not expressed in P. notoginseng.So CYP716 plays a pivotal role in the diversification of ginsenosides in Panax species.Thus, the expression of some differential genes in downstream synthetic pathways in the Panax genus will result in the unique monomer ginsenosides of Panax species.
The CYP716 subgroup is not only hydroxylated at different carbon sites in different members of the Panax genus, but also has different expression levels in different plant tissues (leaf and root tissues).Thus, dammarane-type [synthesized by Y subgroup (CYP716A47) and S subgroup (CYP716A53v2)] and oleananetype [synthesized by A subgroup (CYP716A52v2)] ginsenosides presented different concentrations in leaf and root tissues [91].The CYP716 genes, including PN006374, PN008424, PN011429, and PN029913, show high expression levels in the roots and are likely crucial for the biosynthesis of PPT-type ginsenosides [92,93].

UDP-dependent glycosyltransferases
The UGTs are instrumental in forming various monomeric ginsenosides by attaching monosaccharides to aglycones, primarily at position C-3 or C-20 for PPD-type ginsenosides, and at C-6 and/or C-20 for PPT-type ginsenosides [94].Propanaxanediol and propanaxatriol are glycosylated by various UGT enzymes to form PPD-type and PPT-type ginsenosides, which exist universally in Panax plants.For example PnUGT71A3 is capable of catalyzing both the C6 hydroxyl glycosylation of PPT and F1 into Rh1 and Rg1, and the C20 hydroxyl glycosylation of Rg3 into Rd [95].PnUGT94M1 facilitates the C2 hydroxyl rhamnosylation of Rg1 and Rh1 into Re and Rg2, respectively.PnUGT74A is able to convert CK into F2, PnUGT94C transforms Rh2 into Rg3 and F2 into Rd, and PnUGT71A modifies PPT into F1 and PPD into CK [96].The presence of PnUGT255 is associated with R1 levels, and PnUGT190 significantly inf luences Rd concentrations [97].Elevated expression of three UGT71 genes (PN000453, PN025151, and PN025152) and three UGT74 genes (PN000316, PN024572, and PN033259) in the aerial parts is linked to the biosynthesis of PPD-type ginsenosides [98].

Transcriptional regulation
The biosynthesis and accumulation of ginsenosides are characterized by distinct spatio-temporal patterns.The expression of genes involved in biosynthesis is regulated by a variety of TFs and non-coding RNAs (ncRNAs) through intricate regulatory networks.These TFs and ncRNAs can respond to both biotic and abiotic signals, playing crucial roles in the regulation of ginsenoside production.
Data from the Panax genome and transcriptome database (NCBI accession number PRJNA488357) significantly enhance gene research, although reports on the transcriptional regulatory networks for ginsenoside biosynthesis are scarce.Utilizing multiomics data and patterns of ginsenoside accumulation, coexpression networks within P. notoginseng have been developed to hypothesize the regulatory interactions between TFs and genes involved in ginsenoside biosynthesis (Fig. 4 and Supplementary Data File 2).TFs were categorized using the Plant Transcription Factor Database PlantTFDB (http://planttfdb.gaolab.org/),and Pearson's correlation coefficients were calculated to determine the relationships between TFs and ginsenoside biosynthetic genes using tools from OmicStudio (https://www.omicstudio.cn/tool)[108].Analysis of the P. notoginseng transcriptome database identified 103 TFs that are predicted to have significant positive or negative correlations with the regulation of ginsenoside biosynthetic genes (Pearson's |r| > 0.8, adjusted P < 0.05).This correlation underscores the complexity of the transcriptional regulatory network that mediates ginsenoside biosynthesis.Each biosynthetic gene is inf luenced by multiple TFs, which can either enhance or suppress its activity.Further research is necessary to understand the regulatory functions and interactions among TFs in P. notoginseng to clarify how they control the synthesis of specialized ginsenosides.Additionally, many TFs are capable of simultaneously regulating multiple biosynthetic genes; thus, uncovering the molecular mechanisms that inf luence these transcriptional regulations is crucial for developing innovative breeding strategies.

Non-coding RNAs
In the roots of P. notoginseng, miR156 and miR166 are recognized as the predominant miRNA families, with miR156i and miR156g being the most abundant within these groups [109].The miR156 family, alongside one of its target genes from the squamosa promoter-binding protein-like (SPL) category, exhibits inversely related expression levels that are closely associated with the increase in root biomass content [110].

Environmental regulation
Environmental factors collaboratively inf luence the synthesis of active ingredients through hormone signal-transcription regulatory networks in medicinal plants [111].This environmental regulation encompasses responses to heavy metals, various environmental conditions, and fertilizer applications, as illustrated in Fig. 5.

Heavy metal regulation
The relationship between heavy metals and plant growth, alongside their impact on metabolite synthesis, can be described as 'low promotion and high inhibition' [112].At low concentrations, cadmium (Cd) increases the expression of DS, whereas high concentrations inhibit it; additionally, the presence of Cd significantly reduces the expression of cytochrome P450 enzymes [113].However, the expression levels of DS and P450 genes do not show a strong correlation with the contents of R1, Rb1, Rg1, and total ginsenoside (P > 0.5).Endogenous nitric oxide (NO) boosts the accumulation of Rb1 but lowers the levels of Re and Rg1.Furthermore, endogenous NO increases the transcription of β-amyrin synthase (β-AS), cycloartenol synthase (CAS), and squalene epoxidase (SE), yet decreases the transcription of DS under Cd stress [114].

Environmental factor regulation
Recent studies have demonstrated that environmental factors such as light, temperature, water, and salinity inf luence the synthesis and accumulation of secondary metabolites in medicinal plants [115].Both the rhizosphere and endophytic microf lora of P. notoginseng play a role in enhancing plant health, biomass production, and the synthesis or biotransformation of ginsenosides, either directly or indirectly [116].Climate, soil, and microbial interactions synergistically affect the contents of ginsenosides in P. notoginseng [117].The concentrations of total and individual ginsenosides in the taproot of P. notoginseng show regional variations, with higher ginsenoside levels achievable by increasing temperature in January, atmospheric humidity, and soil calcium content, and by reducing latitude and average July temperatures [118].Additionally, sunshine duration is a significant factor in ginsenoside production, with content increasing alongside longer exposure to sunlight [119].

Fertilizer regulation
Optimal fertilizer application during the cultivation of P. notoginseng enhances both biomass and ginsenoside content [120].The expression of PnbZIP TFs in roots varies under different nitrogen fertilizer conditions; notably, PnbZIP46 is significantly upregulated under ammonium nitrogen fertilizer stress, which may contribute to nitrogen stress resistance [121].Adjusting the nitrogen/potassium application ratio to 1:2 can improve the synergistic effect on both biomass and ginsenoside content in P. notoginseng [122].A lower nitrogen/potassium ratio enhances photosynthesis, sugar accumulation, and the expression of genes involved in ginsenoside biosynthesis.Additionally, the use of ammonium and nitrate fertilizers stimulates the tricarboxylic acid (TCA) cycle, thereby increasing both biomass and ginsenoside content in P. notoginseng roots [123].Conversely, high nitrogen concentrations can inhibit biomass and ginsenoside content; excessive nitrogen reduces root biomass and ginsenoside accumulation, likely due to decreased nitrogen efficiency and reduced photosynthetic capacity [124].

Biotechnological methods for saponin production
Panax notoginseng has a slow growth rate, typically requiring 3-4 years from seed germination to root harvest in field cultivation.Additionally, challenges such as plant pathogens and pests must be managed during the cultivation process.Metabolic engineering offers a viable alternative for enhancing the production of natural products.Consequently, biotechnological approaches, including tissue culture, adventitious roots, transgenic plants, and microbial cell factories, are recommended to boost ginsenoside production (Table 2).

Tissue culture
Suspension culture is an effective method for producing ginsenosides in P. notoginseng.The components of the medium, such as sugar types, significantly impact the ginsenoside concentration in cultured cells.Specifically, 2-hydroxyethyl jasmonate (HEJA) enhances the activity of protopanaxadiol 6-hydroxylase, thereby increasing the contents of Rb1 and Rg1 in cell cultures [125].HEJA is more effective than MeJA in stimulating ginsenoside biosynthesis and altering its composition.Treatment with HEJA results in a 60% increase in total ginsenoside content and a 30% increase in the Rb/Rg ratio [126].Additionally, adding 1 mM phenobarbital to the cultures boosts the levels of PPT-type ginsenosides (Rg1 + Re) [127].Introducing 200 μM MeJA to the cultures can raise the levels of both PPD-type and PPT-type ginsenosides 9-and 2-fold, respectively [128].Plant hormones such as HEJA, endogenous jasmonic acid (JA), and MeJA can induce the upregulation of squalene epoxidase (SE) and the suppression of cycloartenol synthase (CAS), promoting ginsenoside synthesis in P. notoginseng cells [129].
Hairy root and adventitious root cultures have been effectively used to produce stable biomass and high production of ginsenosides in Panax plants for over 15 years [151].Research has revealed that an adventitious root line derived from wild-type roots of P. notoginseng has a high total ginsenoside yield of 17.92 mg/g [130].In P. notoginseng adventitious roots, the highest total ginsenoside content reaches 71.94 mg/g after treatment with 5 mg/l JA, marking an 8.45-fold increase compared with the control [131].Both JA and methyl dihydrojasmonate significantly boost Rd and Rg contents in these roots [131].

Transgenic plants
Despite considerable efforts to produce ginsenosides through tissue and cell culture, the yield remains relatively low.Employing genetic engineering to manipulate gene expression has proven to be an effective strategy for enhancing ginsenoside content.Transgenic P. notoginseng cell lines show higher expression of farnesyl pyrophosphate synthase (FPS) and lower expression of cycloartenol synthase (CAS) compared with wild-type cells, resulting in increased total ginsenoside production and decreased phytosterol levels [132].In transgenic FPS-positive P. notoginseng cell lines, both relative PnFPS expression and ginsenoside contents are significantly higher, with increases of 2.66 times for Rh1, 1.76 times for Rg1, 4.35 times for Re, and 2.90 times for Rd, than in nontransgenic controls [133].Moreover, enhancing β-amyrin synthase (β-AS) expression in transgenic P. notoginseng cells elevates the expression of key enzymes involved in ginsenoside biosynthesis and increases the levels of specific ginsenosides such as chikusetsusaponin IV and IVa [134].Additionally, when three enzyme genes (PnDDS, CYP12H, and UGTPn3) were introduced into tobacco the three exogenous genes were expressed in the roots, stems, and leaves of the transgenic plants, and thus ginsenoside Rh2 and its precursors were successfully synthesized [152].
TFs such as PnbHLH1 and PnERF1 are known to enhance ginsenoside biosynthesis and accumulation.In P. notoginseng cell lines engineered to express PnbHLH1, the total ginsenoside content is more than double that of the control, specifically 2.27 times greater [103].Similarly, in cell lines with PnERF1 transgenic modifications, total ginsenoside content approximately doubles from 40 in control lines to 80 mg/g [106].Additionally, both RNAi targeting PnCAS and overexpression of PnbHLH in P. notoginseng cell lines result in increased levels of total and individual ginsenosides (Rd, Rb1, Re, Rg1, and R1) compared with wild-type and PnCAS RNAi cells [104].

Conclusions and future perspectives
Panax notoginseng and its secondary metabolites are invaluable for human therapy and healthcare, leading to an increased use of this plant as a primary ingredient in numerous products.Consequently, demand for P. notoginseng continues to rise.However, limited varieties, challenges with continuous cropping, and other issues pose significant obstacles to the sustainable development of the P. notoginseng industry.Cultivating new and high-quality varieties is crucial for the ongoing sustainability and growth of this sector.

Modern omics technology accelerates breeding of P. notoginseng
Currently, the affordability and accuracy of high-throughput sequencing have enhanced the feasibility of accessing gene resources.Utilizing high-throughput sequencing technologies and bioinformatics to deeply explore genomic, transcriptomic, and proteomic data of plants aids in the discovery of novel genes.Advanced assays such as quantitative mass spectrometry-based, f luorescence-based, and other high-throughput methods enable rapid detection of enzyme activity and efficient screening of genes.Modern omics technologies are pivotal in accelerating the breeding of new P. notoginseng varieties and providing valuable germplasms for the sustainable development of the P. notoginseng industry.

Transcriptional regulatory networks reveal synthesis mechanisms
TFs play crucial roles in regulating both biotic and abiotic stresses during the growth of P. notoginseng plants, with ginsenoside accumulation closely tied to their regulatory functions and environmental adaptability.Efforts to construct a co-expression network of TFs and gene expression patterns in P. notoginseng have been made; however, the intricate regulatory mechanisms of these TFs require further exploration.Discovering novel genes and TFs will significantly enhance molecular plant breeding.Many TFs involved in ginsenoside production also control multiple genes simultaneously, highlighting the need for a comprehensive understanding of the molecular mechanisms that inf luence transcriptional regulation of ginsenosides to develop innovative breeding strategies.

Synthetic biology increases ginsenoside production
Given that the cultivation of P. notoginseng is both timeconsuming and labor-intensive, the development of bioengineering approaches, such as tissue culture, adventitious roots, transgenic plants, and microbial cell factories, has been pursued to enhance ginsenoside production.Thoroughly understanding the biosynthesis and regulatory mechanisms of ginsenosides will greatly benefit their biotechnological production.These advancements provide an affordable and efficient industrial platform for producing ginsenosides.Identifying new ginsenoside synthesis is expected to pave the way for novel methods that facilitate efficient and large-scale production of ginsenosides.

Figure 1 .
Figure 1.Specialized ginsenosides isolated from P. notoginseng.A Structures of major ginsenoside types.B Relative ginsenoside content distribution in the aerial and underground parts.

Figure 2 .
Figure 2. Summary of pharmacological effects and relevant mechanisms in P. notoginseng.

Figure 4 .
Figure 4. Regulation relationship between TFs and genes involved in ginsenoside synthesis from P. notoginseng.A Regulatory network of CYPs and UGTs with TFs.B Regulatory network of genes from ginsenoside skeleton formation and TFs.A total of 103 TFs were identified, the transcript abundances of which were significantly correlated with functional genes (Pearson's |r| > 0.8, adjusted P < 0.05).Edges represent significant correlations, and node size is mapped to degree.

Table 2 .
Biosynthesis of ginsenosides by metabolic engineering